Characterizing the role of MINK1 in Congenital Heart Disease and mucociliary clearance

项目摘要/摘要 先天性心脏病(CHD)是最常见的出生缺陷，约占所有活产的1% 在美国，它是婴儿死亡的主要原因之一。一种严重的先天性心脏病可由异位症引起 (HTX)，一种胚胎发育过程中左-右(LR)模式的紊乱。一项最新的遗传分析 异质性患者发现了一个新的CHD候选基因mink1。Mink1编码一个丝氨酸-苏氨酸 生发中心激酶在JNK和PCP/Wnt信号通路中的已知功能。然而，它没有 已知在LR构型或心脏发育中的作用。CRISPR基因敲除策略在高吞吐量中的应用 人类疾病模型，非洲爪哇，Mink1的缺失会导致心脏和LR模式缺陷，以及 可移动的纤毛形成和由此产生的流体流动。这项提案的总体目标是调查 Mink1影响LR构型、心脏发育和纤毛形成的分子机制 非洲爪哇(青蛙)模型系统。第一个目标将使用功能损失实验来确定 在测试分子标记的LR图案级联b中，mink1所需的角色。机械论假说 将通过对mink1在整个胚胎和左右胚胎中的时空表达的分析来指导 组织者。第二个目标将使用功能丧失实验来确定对Mink1在 非洲爪哇表皮多纤毛细胞上活动纤毛的形成，概括了一个共同的 冠心病患者存在粘液纤毛缺陷。假设将通过对相关文献的回顾来指导 包括通过Notch信号在调节多纤毛细胞命运规范中的作用或要求 Mink1在基础身体对接和/或建立极性期间。第三个目标将决定每个目标的作用 Mink1结构域在多纤毛细胞上纤毛形成和LR图案化/心脏发育的背景下。 然后，将确定患者在激酶编码区域的突变是否是决定发挥作用 在非洲爪哇中使用多功能分析。总之，这些实验将提高我们对 心脏发育及mink1在冠心病发病机制中的作用。在未来，这将有利于基因 检测和咨询，以及改善CHD的结果，因为治疗可以针对基因定制 而不是仅仅根据CHD的表型。此外，这份申请书详细说明了申请人的培训计划，包括 研究指导、高级课程、新技术培训和技能发展 科学的专业性，数据的写作和表达。其中概述的研究和培训 申请者将做好准备，以独立的身份从事以患者为导向的研究 研究科学家。

Project Summary/ Abstract Congenital Heart Disease (CHD) is the most common birth defect affecting approximately 1% of all live births in the US and is one of the leading causes of infant mortality. A severe form of CHD can result from Heterotaxy (Htx), a disorder of the left-right (LR) patterning during embryonic development. A recent genetic analysis of heterotaxy patients identified a novel CHD candidate gene, mink1. Mink1 encodes a serine-threonine germinal-center kinase with known functions in the JNK and PCP/Wnt signaling pathways. However, it has no known role in LR patterning or cardiac development. Using CRISPR knockout strategies in the high-throughput human disease model, Xenopus, loss of mink1 leads to cardiac and LR patterning defects, and defects in motile cilia formation and resulting fluid flow. The overall goal of this proposal is to investigate the molecular mechanism by which mink1 affects LR patterning, heart development, and cilia formation in the Xenopus (frog) model system. The first aim will use loss of function experiments to determine the required role for mink1 during the LR patterning cascade b testing molecular markers. Mechanistic hypotheses will be guided by an analysis of temporal and spatial expression of mink1 in the whole embryo and the left-right organizer. The second aim will use loss of function experiments to determine the requirement for mink1 during formation of motile cilia on the multi-ciliated cells of the Xenopus epidermis, which recapitulates a common mucociliary defect found in CHD patients. Hypotheses will be directed by a review of relevant literature including a role in regulation of multi-ciliated cell fate specification through Notch signaling or a requirement for Mink1 during basal body docking and/or establishment of polarity. The third aim will determine the role of each mink1 domain in the context of cilia formation on multi-ciliated cells and LR patterning/cardiac development. Then it will be determined if the patient mutation in the kinase-encoding domain is determination to function using multiple functional assays in Xenopus. Altogether, these experiments will improve our understanding of cardiac development and the role of mink1 in the pathogenesis of CHD. In the future, this will benefit genetic testing and counseling, as well as improve outcomes in CHD because treatments can be tailored to genotype rather than solely on CHD phenotype. In addition, this application details the applicant's training plan including research mentorship, advanced coursework, training in new techniques, and the development of skills in scientific professionalism, writing, and presentation of data. The research and training outlined in this application will prepare the applicant to pursue a career performing patient-driven research as an independent research scientist.